Pelletier Amélie, Joly Erik, Prentki Marc, Coderre Lise
Montreal Diabetes Research Centre, Centre Hospitalier de l'Université de Montréal and the Department of Medicine, Université de Montréal, Montréal, Québec, Canada H2W 1T7.
Endocrinology. 2005 May;146(5):2285-94. doi: 10.1210/en.2004-1565. Epub 2005 Jan 27.
During metabolic stress, such as ischemia or hypoxia, glucose becomes the principal energy source for the heart. It has been shown that increased cardiac glucose uptake during metabolic stress has a protective effect on cell survival and heart function. Despite its physiological importance, only limited data are available on the molecular mechanisms regulating glucose uptake under these conditions. We used 2,4-dinitrophenol (DNP), an uncoupler of oxidative phosphorylation, as a model to mimic hypoxia and gain insight into the signaling pathway underlying metabolic stress-induced glucose uptake in primary cultures of rat adult cardiomyocytes. The results demonstrate that 0.1 mM DNP induces 2.2- and 9-fold increases in AMP-activated protein kinase (AMPK) and p38 MAPK phosphorylation, respectively. This is associated with a 2.3-fold increase in glucose uptake in these cells. To further delineate the role of AMPK in the regulation of glucose uptake, we used two complementary approaches: pharmacological inhibition of the enzyme with adenine 9-beta-D arabinofuranoside and adenoviral infection with a dominant-negative AMPK (DN-AMPK) mutant. Our results show that overexpression of DN-AMPK completely suppressed DNP-mediated phosphorylation of acetyl coenzyme A carboxylase, a downstream target of AMPK. Inhibition of AMPK with either 9-beta-D arabinofuranoside or DN-AMPK also abolished DNP-mediated p38 MAPK phosphorylation. Importantly, AMPK inhibition only partially decreased DNP-stimulated glucose uptake in cardiomyocytes. Inhibition of p38 MAPK with the pharmacological agent PD169316 also partially reduced (70%) glucose uptake in response to DNP. In conclusion, our results indicate that p38 MAPK acts downstream of AMPK in cardiomyocytes and that activation of the AMPK/p38 MAPK signaling cascade is essential for maximal stimulation of glucose uptake in response to DNP in adult cardiomyocytes.
在代谢应激状态下，如缺血或缺氧时，葡萄糖成为心脏的主要能量来源。研究表明，代谢应激期间心脏葡萄糖摄取增加对细胞存活和心脏功能具有保护作用。尽管其具有生理重要性，但关于在这些条件下调节葡萄糖摄取的分子机制的可用数据有限。我们使用2,4-二硝基苯酚（DNP），一种氧化磷酸化解偶联剂，作为模拟缺氧的模型，以深入了解成年大鼠心肌细胞原代培养物中代谢应激诱导的葡萄糖摄取的信号通路。结果表明，0.1 mM DNP分别使AMP活化蛋白激酶（AMPK）和p38丝裂原活化蛋白激酶（p38 MAPK）磷酸化增加2.2倍和9倍。这与这些细胞中葡萄糖摄取增加2.3倍相关。为了进一步阐明AMPK在葡萄糖摄取调节中的作用，我们使用了两种互补方法：用腺嘌呤9-β-D阿拉伯呋喃糖苷对该酶进行药理学抑制以及用显性负性AMPK（DN-AMPK）突变体进行腺病毒感染。我们的结果表明，DN-AMPK的过表达完全抑制了DNP介导的乙酰辅酶A羧化酶（AMPK的下游靶点）的磷酸化。用9-β-D阿拉伯呋喃糖苷或DN-AMPK抑制AMPK也消除了DNP介导的p38 MAPK磷酸化。重要的是，AMPK抑制仅部分降低了心肌细胞中DNP刺激的葡萄糖摄取。用药物PD169316抑制p38 MAPK也部分降低了（70%）对DNP的葡萄糖摄取反应。总之，我们的结果表明，p38 MAPK在心肌细胞中位于AMPK下游，并且AMPK/p38 MAPK信号级联的激活对于成年心肌细胞中对DNP的葡萄糖摄取的最大刺激至关重要。
